Biophysical Journal: Biophysical Letters
Second Harmonic Generation in Neurons: Electro-Optic Mechanism
of Membrane Potential Sensitivity
Jiang Jiang,* Kenneth B. Eisenthal,y and Rafael Yuste*
*Howard Hughes Medical Institute, Department of Biological Sciences, and yDepartment of Chemistry, Columbia University, New York
ABSTRACT Second harmonic generation (SHG) from membrane-bound chromophores can be used to image membrane potential
in neurons. We investigate the biophysical mechanism responsible for the SHG voltage sensitivity of the styryl dye FM 4-64 in pyramidal
neurons from mouse neocortical slices. SHG signals are exquisitely sensitive to the polarization of the incident laser light. Using this
polarization sensitivity in two complementary approaches, we estimate a ;36 tilt angle of the chromophore to the membrane normal.
Changes in membrane potential do not affect the polarization of the SHG signal. The voltage response of FM 4-64 is faster than 1 ms
and does not reverse sign when imaged at either side of its absorption peak. We conclude that FM 4-64 senses membrane potential
through an electro-optic mechanism, without significant chromophore membrane reorientation, redistribution, or spectral shift.
Received for publication 18 April 2007 and in final form 21 June 2007.
Address reprint requests and inquiries to R. Juste or K. B. Eisenthal, E-mail: rmy5@columbia.edu; kbe1@columbia.edu.
Jiang Jiang’s present address is Institute of Bioengineering and Nanotechnology, Singapore.
Traditionally, the membrane potential of neurons is measured with intracellular electrical recordings or patch-clamping.
Although these techniques have been, and continue to be,
powerful methods in neuroscience, they cannot be used to
monitor membrane potential at dendritic spines, which are the
predominant sites of excitatory inputs into a neuron. The reason
is that spines are too small (;1 mm3) to accommodate an
electrode. A promising nonlinear optical technique, second
harmonic generation (SHG) (1–3), can be used to measure the
electrical potential at various interfaces, e.g., charged surfactant/water (4) and charged phospholipid liposome/water interfaces (5). SHG has been recently used to measure membrane
potential of biological cells in an imaging microscopy mode,
using a variety of chromophores that are adsorbed to the cell
membrane (3).
We recently carried out the first membrane potential measurements of individual spines using FM 4-64 (6), a fluorophore used to study vesicle recycling (7), which is also an
excellent SHG chromophore (8,9). We observed a linear
dependency between SHG signals and membrane potential,
with a sign reversal at zero voltage, and proposed that the
voltage response of FM 4-64 SHG was likely electro-optic
(6). However, other mechanisms of voltage sensing, such as
chromophore redistribution, reorientation, or spectral shift
(10–12), could not be ruled out.
We now explore the biophysical mechanisms of FM 4-64
SHG voltage sensitivity, characterizing its polarization, wavelength dependence and the speed of its response, to better
understand how the chromophores behave in response to
changes in electric field.
Most push-pull styryl dyes, such as FM 4-64, possess a
long molecular axis, and their hyperpolarizability tensor is
dominated by a single tensor element along that axis (10,11,13–
16). With this simplification and the assumption that the orienBiophysical Journal: Biophysical Letters
tation of the dye in the plane of the membrane is isotropic and
peaked at a fixed angle, the tilt angle of the dye molecule with
respect to the plasma membrane can be analytically determined.
Following past work on artificial membranes (10), the intensity
of SHG versus the angle f between excitation light polarization
and the membrane normal can be expressed as
3
2
2
2
2
SHG ¼ SHG0 f½Æcos uæcos f 1 Æcosu sin uæsin f=2
1 Æcosu sin uæ sin f cos fg;
2
2
2
2
(1)
whereby SHG0 is a constant, u the angle of dye axis to membrane normal, and Æ. . .æ denotes ensemble average of dye
molecules with different orientations (Fig. 1 A).
We performed SHG measurements from layer-5 pyramidal
neurons in neocortical brain slices, filled with FM 4-64 through
a patch pipette and held at 65 mV. Due to the dominant
uniaxial hyperpolarizability of FM 4-64, various parts of the
plasma membrane showed strong SHG signals depending on
the relative angle of the laser polarization and the local membrane, with orthogonal segments of the membrane showing
negligible SHG signals (Fig. 1 B). Because neurons are not
perfectly spherical, we sampled a small region of the soma and
varied the polarization of the excitation light. The value u was
then obtained from cosu ¼ 0.81 6 0.02 (mean 6 SD; n ¼ 5
neurons) by fitting the data using Eq. 1 (Fig. 2), yielding an
average dye tilt angle of ;36 to the membrane normal,
assuming a narrow orientational distribution.
As an alternative approach to measure dye orientation, we
analyzed the polarization of the SHG signal. By separating
the SHG components parallel and perpendicular to the same
Editor: Barry R. Lentz.
2007 by the Biophysical Society
doi: 10.1529/biophysj.107.111021
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Biophysical Journal: Biophysical Letters
excitation polarization, we also found an average dye tilt-angle
of ;36 (n ¼ 2, see Supplementary Material Fig. S1). Thus,
two complementary approaches yielded the same estimation.
We then investigated whether the dye orientation could be
altered by changes in the local electric field. For these experiments, we clamped the membrane potential of the neurons at
voltages ranging from 95 mV to 0 mV, while SHG images of
the cell were recorded at different laser polarizations (Fig. 3).
No dye tilt-angle change was observed within our detection
limit, even for membrane potential changes as large as 90 mV
(or a change of electric field of 1.6 3 107 V/m, assuming
membrane thickness of 5 nm). These results were reliably
reproduced across neurons (n ¼ 3). Using Eq. 1, we calculated
a confidence interval for our dye tilt-angle measurement. As
shown in Fig. 3, a 5 change in dye tilt-angle would result in a
significant change in the shape of fitted curve.
The observed insensitivity of dye orientation to the transmembrane field is intriguing, as the change of electric field
applied was on the order of tens of MV/m. One possibility is
that there is a broad distribution of tilt-angles (17), which
would not be sensitive to membrane potential. Another possibility is that the local electrostatic fields, e.g., dipolar, steric
forces imposed by membrane phospholipids chains and other
membrane biomolecules, or hydrophobic and hydrophilic
forces, are the dominant orientational factors in the membranes of the neurons used in this study.
The voltage sensitivity of fluorescent probes can be due to
a change in the molecular electronic transition (12), reorientation of the membrane-bound dye (18), or a voltage-dependent
slow redistribution of the population of dye molecules (19).
Unfortunately, studies of the mechanisms of electric field
sensing by SHG chromophores are still scarce. In artificial
membranes consisting of one type of phospholipid, two
mechanisms of SHG electric field sensing were described: a
purely electro-optical response due to a third-order polarization
with one of the fields being static (10), and a combination of
electro-optical and fast chromophore reorientation (11). In
addition, a very slow (35–200 ms) SHG response to membrane
potential changes has been reported (20), incompatible with an
fast electro-optic mechanism.
To characterize the speed of the FM 4-64 response to voltage
we performed fast (0.1 ms resolution) measurements of somatic
membrane regions of neurons, under conditions where action
potentials were generated by brief current injections (Fig. 4).
In these experiments, the temporal response of FM 4-64 to a
change in the electric field was instrument-limited, on the order
of a microsecond timescale. As demonstrated in Fig. 4, the
FIGURE 2 SHG power versus angle of laser polarization (mean 6
SE, n 5 5 neurons) . Line is a nonlinear fit, based on Eq. 1.
FIGURE 3 Polarization dependence of SHG signal at different
membrane potentials. Solid curve is the best fit with u 5 38; dotted
lines are fitted curve with u 5 43 and u 5 33, respectively.
FIGURE 1 (A) Simplified representation of FM 4-64 geometry
in membrane. Arrow shows average orientation of the uniaxial
hyperpolarizability, with u being its tilt-angle to membrane normal.
(B) Membrane SHG signals polarization dependence; arrow indicates laser polarization.
Biophysical Journal: Biophysical Letters
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Biophysical Journal: Biophysical Letters
REFERENCES and FOOTNOTES
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FIGURE 4 Fast kinetics of action potential SHG measurements.
Relative change in SHG (blue line) shows similar kinetics as the
electrical signal, recorded with a whole-cell electrode (dashed
black line). Note the linearity of the optical response to voltage
and how the peaks coincide.
SHG signal from FM 4-64 tracked the fast time course of the
action potential, without any appreciable delay.
A fast SHG response to the electric field could be due to a
Stark shift in the spectrum, and a telltale sign of this would
be that the SHG relative change would switch signs as the
SHG frequency moves through the absorption peak. In
agreement with other styryl dyes measurements (20), we did
not observed a change in sign when imaging FM 4-64 SHG
on either side of its absorption peak (;500 nm). Specifically,
the change in SHG in response to a 100-mV depolarization
was 10.3 6 0.8% at 850 nm (n ¼ 15), 10.6 6 1.1% at 900
nm (n ¼ 13), and 14.8 6 1.2% at 1064 nm (n ¼ 19).
We conclude that the potential sensing mechanism of FM
4-64 in neurons is predominantly electro-optical. No physical
reorientation of the dye was detected upon changing membrane
potential and the response was as fast as our time resolution and
had no wavelength dependency in its sign. Finally, the similar
linearity of the response at slow (6) and fast (Fig. 4) temporal
resolutions indicates that the mechanisms of voltage sensitivity
are likely to be the same at these two different timescales.
Although the exact mechanism of SHG potential sensing is not
critical as long as the response is fast, linear, and calibrated
before any quantitative interpretation, its understanding is
important for improving probe design, a crucial issue given that
SHG measurements of fast voltage transients in spines and
dendrites are currently limited by poor signal/noise ratio (6,21).
Chromophores that combine strong electro-optic and molecular orientation response (11) would be ideal for optimal SHG
potential imaging with high sensitivity.
SUPPLEMENTARY MATERIAL
To view all of the supplemental files associated with this
article, visit www.biophysj.org.
ACKNOWLEDGMENTS
Supported by the NYSTAR program, the Kavli Foundation, the Gatsby
Institute for Brain Science and the Binational US-Israel Science Foundation. K.B.E. thanks the National Science Foundation for their support.
Biophysical Journal: Biophysical Letters
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